Drosophila Nhe2 overexpression induces autophagic cell death

Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.

Major. 1) pHi increase is causative of autophagy.This is the thesis of the MS based on the results of Grillo-Hill et al. eLife 2015;4:e03270. (DOI: 10.7554/eLife.03270).As noted below, I believe there are short-comings in that paper, but here I will focus on the causal link of alkalinization causes autophagy.Simply put, this MS does not test that thesis a. as noted below, there are NO pHi measurements in the MS, making it impossible to establish a causal link b.Authors have not attempted to manipulate pHi with non-specific targeting of the fly eye to determine if pHi increase itself elicits this response c.Authors do not know what genetic manipulations in Figures 3 and 4 do to pHi.This becomes crucial to support their current thesis as Figure 3 implicated apoptosis (autophagy) based on the partial ocular phenotype of H99 in the background of GMR>DNhe2 vs. GMRGAL4 x UAS-H99.We do not know IF H99 manipulation changes pHi or just the morphologic phenotype.While I agree it is likely, it is not demonstrated experimentally.d. Discussion of H99 effected (downstream genes: grim, rpr and hid) would seem to be needed.Also note that only hid is even detectable in FlyAtlas [~2 FPKM in eye].It is unclear what this might mean for the overall pathway stressed by the authors.e.Similarly, with UAS-p35 crosses, we do not know that the lack of a morphologic phenotype is due to no or insignificant pHi changes.f.Fig 4 -more documentation and pHi measurements are needed.According to FlyAtlas (1 or2), the levels of Atg1 (modest, 15 FPKM), Atg7 (low, 4-5 FPKM) and Atg8a (high, ~210) are not remotely similar in the larval brain or adult eye.In this context, it seems unclear why the modest expression of Atg1(3) has the most obvious morphologic phenotype.At minimum, the authors need to explain this.
2) pHi assessment a. (p5, beginning of Results/Discussion) states: "To determine how increased pHi impacts tissue size in the Drosophila eye, quantitative analysis of the area of adult eyes was performed."However, according to the Methods, pHi measurements were performed in this study.It should also be noted that pHi measurements in the eLife 2015 paper, were not properly calibrated, i.e., most of the GMRGAL4>DNhe2 measurements and calculated pHi's are above the top end of their pH 7.5 range.This means that while pHi is almost certainly higher, we do not really know what the pHi range is.This becomes important because almost any animal cell type will overtly die if resting pHi is chronically >7.8.b.Similarly Fig 2 title claims "...with higher pHi" c.If the authors merely elevate pHi chemically (e.g., chronic NH4Cl or similar), what happens?This approach should distinguish between specific vs. generalized cell death 3) Cell Count assessment a.How are the 3 different counts of cells in any given experiment handled for "N" total number of cells?b.Are the 3 different "readers" always the same for each reported experiment?c.Some sampling of reader1, reader2 and reader3 should be included as we do not know how these counts differ (could be as supplemental), i.e., what is "observer bias?" 4) Imaging details a. Presumably the Zeiss LSM 700 confocal microscope is connected to a monochrome camera, but the type of camera should be specified as indicated in "high-resolution images of Drosophila eyes."b.Unclear that normalizing to Hoechst 33342 channel is appropriate.Authors should explain why that this is done and provide some evidence that is biologically meaningful.Have the authors maintained individual channel settings for both photograph and images used for analysis?Even if the above normalization is shown to be appropriate, changing setting at all during imaging for the various channels could lead to artifacts.This is all "pre-FIJI" analysis that should be controlled.• Be consistent with Drosophila genetics and phenotypes.We do not need the detail found in Methods, but the non-fly audience needs to be able to understand the drivers (Gal4 line(s)) and targets (UAS lines).For example, GMRGAL4 would typically be GMR(italics)-Gal4 and UAS as UAS-H99, with the F1 cross GMR>H99, etc.We are submitting a revised manuscript now titled "Drosophila Nhe2 Overexpression Induces Autophagic Cell Death".We thank the reviewers for their time reviewing this work, and their constructive comments and suggestions to improve our manuscript.Reviewer 2 praised it as "a new and significant finding" and said "the data and conclusions are convincing."Reviewer 1 "tends to agree that the autophagy pathway is in play".We are glad that both reviewers support resubmission with minor revisions.
We are submitting a revised manuscript that adds significant points of clarification and additional information based on reviewer comments and suggestions.Our revision includes editorial changes to address Reviewer 1's concerns about the conflation of increased intracellular pH with DNhe2 over-expression.We added rationale to the introduction, and restricted discussion of increased pHi as the probable mechanism to the introduction and discussion sections.We also addressed Reviewer 2's comments regarding our figures, and revised and standardized our notations and labels to better communicate genotypes, statistical significance, and added additional detail to figure legends.
Our revision addresses all reviewer concerns, and below we include a point-bypoint response to each reviewer concern.We believe that this is a stronger submission and hope that it is suitable for publication in Molecular Biology of the Cell.

Point-by-point responses to reviewer comments:
Reviewer #1: General.The current MS uses Drosophila genetics and eye morphology and cell biology to support their thesis that increased intracellular pH (increased pHi, i.e., alkalinization) is the causal driver of this through the autophagy pathway.This reviewer would tend to agree that the autophagy pathway is in play; however, the explicit link to alkalinization or even altered pHi is not demonstrated.Key experiments to demonstrate cause and effect are needed (noted below).Either this link should be made or the MS recast as a more cell biology oriented MS.
We thank this reviewer for their thorough review of our manuscript.Their comments helped us identify areas of the text that required clarification and improved explanation, and we appreciate their time and work in improving our manuscript.Overall, we appreciate their comment regarding the explicit link between autophagy and increased pHi.We have revised the introduction to emphasize our rationale, including discussing in detail data from our previous publications that suggests that increased pHi through DNhe2 over-expression is responsible for the rough eye phenotype.We also edited the text to throughout the results section to clarify that over-expression of DNhe2 (rather than increased pHi) is the experimental manipulation used.Finally, we restricted discussion of increased pHi as the probable mechanism (based on our data and previously published data) to both the introduction and discussion sections.Grillo-Hill et al. eLife 2015;4:e03270. (DOI: 10.7554/eLife.03270).As noted below, I believe there are short-comings in that paper, but here I will focus on the causal link of alkalinization causes autophagy.Simply put, this MS does not test that thesis a. as noted below, there are NO pHi measurements in the MS, making it impossible to establish a causal link

Major. 1) pHi increase is causative of autophagy. This is the thesis of the MS based on the results of
We thank this reviewer for their comment, and we have made revisions throughout the manuscript to address this concern.In our eLife 2015 paper, we showed that the retinal patterning errors that we observe with over-expression of DNhe2 are in fact due to the ion transport function of the protein, rather than one of its other functions.Specifically, using the mammalian transport-dead E266I NHE1 mutation as a template, we identified the orthologous residue in Drosophila DNhe2 (E358I), and generated transgenic flies carrying this predicted "transport dead" mutant.We found that when we overexpressed the transport dead mutant, DNhe2 E358I , there was no change in pHi and no visible phenotypes in the Drosophila eye.We have revised the introduction (p. 3, 4) to emphasize this rationale, the prior results that show rough eye phenotype arises from ion transport activity of DNhe2, and to clarify that over-expression of DNhe2 is the experimental manipulation we use throughout the results section (p.6, 7).We restricted discussion of increased pHi as the probable mechanism to the introduction and discussion sections.

b. Authors have not attempted to manipulate pHi with non-specific targeting of the fly eye to determine if pHi increase itself elicits this response
Thank you for the suggestion.We agree that the proposed experiment would strengthen the causal connection between increased pHi and autophagy phenotypes.However, we lack alternative experimental approaches to robustly manipulate pHi in vivo in Drosophila.In addition to DNhe2 overexpression (used in this manuscript), only one other tool has been shown to alter pHi in vivo, which is RNAi-mediated knockdown of Drosophila anion exchanger 2 in the Drosophila ovary (Benitez, et.al. 2019).There are not any published methods to non-specifically increase pHi in Drosophila over a sufficiently long time period to evaluate molecular markers of autophagy (often reported to take at least several hours; doi: 10.3390/toxics11080682 and DOI:https://doi.org/10.1016/j.devcel.2004.07.005).Our unpublished experiments with growing Drosophila on NH4Cl-containing media or incubating Drosophila retinae or cell lines in NH4Cl did not result in altered pHi. 3 and 4 do to pHi.This becomes crucial to support their current thesis as Figure 3 implicated apoptosis (autophagy) based on the partial ocular phenotype of H99 in the background of GMR>DNhe2 vs. GMRGAL4 x UAS-H99.We do not know IF H99 manipulation changes pHi or just the morphologic phenotype.While I agree it is likely, it is not demonstrated experimentally.

c. Authors do not know what genetic manipulations in Figures
We thank the reviewer for this comment.We did not perform pHi measurements in this work as we lack access to live-cell-imaging microscopy resources that enable these experiments.Our measurement in these experiments was cell number in pupal eyes, which is a well-established system for studying cell death.We clarified that over-expression of DNhe2 is the experimental manipulation (p.6), and restricted discussion of increased pHi as the probable mechanism to the introduction and discussion sections of the revised manuscript.
d. Discussion of H99 effected (downstream genes: grim, rpr and hid) would seem to be needed.Also note that only hid is even detectable in FlyAtlas [~2 FPKM in eye].It is unclear what this might mean for the overall pathway stressed by the authors.
We thank the reviewer for raising this point, and we expanded our discussion of the H99 deletion in the manuscript (p. 6).We used H99 in our work because it has historically been used to completely block retinal apoptotic cell death induced by genes including Dronc (DOI 10.1074/jbc.M002935200), DIAP1 (doi.org/10.1038/sj.cdd.4401538) and argos (doi.org/10.1038/sj.cdd.4400398).Together with our other data, we interpret this to mean that blocking apoptosis does not fully suppress the morphological defects caused by over-expression of DNhe2.e.Similarly, with UAS-p35 crosses, we do not know that the lack of a morphologic phenotype is due to no or insignificant pHi changes.
We thank the reviewer for raising this point, and again answer that our measurement in these experiments was cell number, and not pHi.We believe that p35 over-expression is not likely to alter pHi, as it has a well characterized mechanism of covalent binding directly to caspases within the active site, thus preventing target protein cleavage (DOI: 10.1038/35068604).There are no published reports of altered pHi with caspase inhibition.
f. Fig 4 -more documentation and pHi measurements are needed.According to FlyAtlas (1 or2), the levels of Atg1 (modest,15 FPKM),Atg7 (low,(4)(5) and Atg8a (high, ~210) are not remotely similar in the larval brain or adult eye.In this context, it seems unclear why the modest expression of Atg1(3) has the most obvious morphologic phenotype.At minimum, the authors need to explain this.
We thank the reviewer for pointing out the FlyAtlas expression data.It is important to note that the limited time points and tissue types contained in the FlyAtlas (retinal tissue only at adult stages) do not overlap with our data, which show missing cells at larval and pupal stages of retinal development.It is also worth noting that transcript levels often correlate poorly with protein levels (doi: 10.1039/b908315d), and for many proteins, abundance and activity are not directly correlated due to post-translational modification, allosteric regulation and environmental factors including pHi.We chose to quantify cell number in our Atg1 3 heterozygotes as these effects were the most robust and reproducible.We cannot rule out that this may be due to variable knock-down in the Atg7 or Atg8a RNAi lines.
2) pHi assessment a. (p5, beginning of Results/Discussion) states: "To determine how increased pHi impacts tissue size in the Drosophila eye, quantitative analysis of the area of adult eyes was performed."However, according to the Methods, pHi measurements were performed in this study.It should also be noted that pHi measurements in the eLife 2015 paper, were not properly calibrated, i.e., most of the GMRGAL4>DNhe2 measurements and calculated pHi's are above the top end of their pH 7.5 range.This means that while pHi is almost certainly higher, we do not really know what the pHi range is.This becomes important because almost any animal cell type will overtly die if resting pHi is chronically >7.8.
In reference to the comment "pHi measurements were performed in this study", we do not in fact perform pHi measurements in the current study.We have revised the manuscript to clarify that over-expression of DNhe2 is the experimental manipulation, and restricted discussion of increased pHi as the proposed molecular driver to the introduction and discussion sections.
The critique in the rest of this section is outside the scope of the current paper, as it critiques previously peer-reviewed and published work that is not included in the current manuscript.Furthermore, we are in agreement with the reviewer's overall conclusion that the 2015 eLIFE paper showed that over-expression of DNhe2 increases intracellular pH in the fly eye.
Finally, we wish to reinforce a point the reviewer makes in the final point in this section that "almost any animal cell type will overtly die if resting pHi is chronically >7.8".We agree that this is a long-standing observation with incompletely characterized molecular mechanism for the mode of death.Critically, in our current study, our results suggest that cell death with prolonged increased pHi acts specifically through the autophagy pathway.

b. Similarly Fig 2 title claims "...with higher pHi"
We have revised the manuscript to clarify that over-expression of DNhe2 is the experimental manipulation, and restricted discussion of increased pHi to the introduction and discussion sections.

c. If the authors merely elevate pHi chemically (e.g., chronic NH4Cl or similar), what happens? This approach should distinguish between specific vs. generalized cell death
As stated above in response to critique 1b, only two manipulations have been shown to increased pHi in Drosophila in vivo: DNhe2 overexpression (used in this manuscript), and RNAi-mediated knock-down of Drosophila anion exchanger 2 in the Drosophila ovary (Benitez, et.al. 2019).There are not any published methods to chemically increase pHi in Drosophila retinal tissue, and our attempts to develop such methods have so far been unsuccessful (unpublished data).
To the second point, the reviewer also makes a distinction between "specific vs. generalized cell death".Unfortunately, without defining what those differences are expected to be, it was impossible to determine what the reviewer means by "specific" and "generalized".Importantly, we used morphological analysis, genetic interaction studies, and genetically-encoded autophagy markers to determine that the observed cell death required proteins in the autophagy pathway.Taken together, our work suggests a cellular mechanism by which overexpression of DNhe2 in the Drosophila eye leads to a cell death phenotype mediated by autophagy pathway members.By definition, this is a specific and molecularly defined cell death pathway (autophagy-mediated).

3) Cell Count assessment a. How are the 3 different counts of cells in any given experiment handled for "N" total number of cells?
We thank the reviewer for this question, and have updated our methods section to reflect our practices (p.10).After image acquisition and processing, at least three individuals perform counts, which are reconciled through discussion into a consensus cell count.All students were trained based on the following protocol: "Cell counts are performed by identifying a central ommatidium and its six nearest neighbors, and drawing a hexagon through the center of the cone cells in the six neighbors.Each cell completely contained in this hexagon counts as one cell, and cells that are partially contained count as one-half cell."Individuals new to cell counting were given previously-analyzed datasets to confirm data concordance with experienced counters.Cell counts were performed by at least 3 different people for reproducibility, and any discrepancies were discussed to consensus.One individual eye imaginal disc or pupal eye are taken from each animal.
b. Are the 3 different "readers" always the same for each reported experiment?
We thank the reviewer for this query.We are a team of scientists at a PUI where the average lab tenure is 1-2 years.The data collection for this manuscript took more than 6 years, so the 3 readers were not always the same across data sets.The PI/corresponding author trains students, spot-checks data for new counters and participates in all data collection and analysis, which provides strong consistency across analyzed datasets.
c. Some sampling of reader1, reader2 and reader3 should be included as we do not know how these counts differ (could be as supplemental), i.e., what is "observer bias?" We thank the reviewer for pointing out this, and have expanded our methods to better explain how we determine counts (p.10).In short, there is no discrepancy in counts to report as the rare cell count differences are discussed to consensus with input and guidance from the PI/corresponding author (see 3a and b responses above).

4) Imaging details
a. Presumably the Zeiss LSM 700 confocal microscope is connected to a monochrome camera, but the type of camera should be specified as indicated in "high-resolution images of Drosophila eyes." We appreciate the reviewer's attention to detail regarding our microscopy methodology.The Zeiss LSM 700 confocal microscope is a ready-built (not modular) system, and the only information provided about the camera is "High-resolution AxioCam microscope camera".We have updated the methods section to include this information.
b. Unclear that normalizing to Hoechst 33342 channel is appropriate.Authors should explain why that this is done and provide some evidence that is biologically meaningful.Have the authors maintained individual channel settings for both photograph and images used for analysis?Even if the above normalization is shown to be appropriate, changing setting at all during imaging for the various channels could lead to artifacts.This is all "pre-FIJI" analysis that should be controlled.
We appreciate this reviewer's thoughtful comments regarding our image acquisition parameters.We have added detail to the methods section to outline our approach (p.10), and provide context and justification for our normalization to Hoechst.We have also edited the methods to explicitly described that control and experimental images were acquired on the same day using identical acquisition settings (p.10), which avoids the artifacts described by the reviewer.

5) Figure Notes a. Fig 3 C should include all the groups in Fig 3A
We appreciate this suggestion, and have added additional text to the results to explain our rationale for only performing counts for the p35 -DNhe2 genetic interaction.After we ruled out caspase involvement in the observed cell death, we chose to focus on identifying the mode of cell death as described in revised Figure 4. Minor.
• Be consistent with Drosophila genetics and phenotypes.We do not need the detail found in Methods, but the non-fly audience needs to be able to understand the drivers (Gal4 line(s)) and targets (UAS lines).For example, GMRGAL4 would typically be GMR(italics)-Gal4 and UAS as UAS-H99, with the F1 cross GMR>H99, etc. Please standardize throughout We appreciate the reviewer's comments and attention to detail.We have standardized our nomenclature throughout the revised manuscript.

SUMMARY
The study by Peralta and colleagues investigates the underlying mechanisms of how changes in intracellular pH (pHi) can impact cell signaling, growth and differentiation of tissues, using development of the Drosophila eye as a genetically tractable model system.Patterning of the Drosophila eye (composed of 750 identical multicellular units called ommatidia) involves sequential phases of cell proliferation (larval), differentiation and regulated cell death (pupal) to achieve the adult eye configuration.Previous studies from this group (Grillo-Hill, B. K., et al. (2015) Elife) and others (Simons, M., et al. (2009).Nat.Cell Biol.) have identified the plasma membrane Na+/H+exchanger Dnhe2 as a critical component of this process, as dysregulation of Dnhe2 disrupts Wnt signaling, planar cell polarity and tissue architecture of the Drosophila eye.
Notably, earlier results showed that overexpression of Dnhe2 caused alkalinization of cells within the eye imaginal disc and induced tissue patterning errors in the ommatidia that manifested visibly as a rough eye phenotype in adult flies.This was associated with increased cell proliferation in the posterior region of the developing eye imaginal disc, yet unexpectedly resulted in decreased overall size of adult eyes.However, the underlying mechanisms were unresolved.In the present study, the authors found that while cone cells were unaffected, there were significant decreases in secondary and tertiary pigmented cells as well as bristle complexes of the ommatidia.Additional evidence (genetic manipulation of anti-apoptotic genes) excluded apoptosis as a contributing factor, and instead identified a previously unrecognized role for genes involved in autophagic cell death.Overall, this is a new and significant finding.The data and conclusions are convincing, though there are several minor concerns in the manuscript that need to be addressed as outlined below.

COMMENTS
The manuscript is generally well written and the interpretations are consistent with the data, although the concluding statement that their findings support "inhibiting autophagy as a therapeutic approach to cancer" is a bit of a stretch since cancerous cells were not examined in this study.Also, the manuscript was not carefully proof-read and contains several errors and omissions that need correcting.
1.There are no page numbers.
We thank this reviewer for pointing out this oversight, and added page numbers to our manuscript.

Page 3 (Introduction)
, it is stated that Drosophila Dnhe2 is the ortholog of the ubiquitous plasma membrane NHE1 isoform in mammals.However, this assertion conflicts with a previous study (Simons, M., et al. (2009).Nat.Cell Biol.) which claims-based on sequence alignments-that DNhe2 is the ortholog of the mammalian epithelial apical membrane NHE3 isoform.Please clarify.
We thank the reviewer for this question.We addressed this discrepancy with the 2009 Nat.Cell Biol paper in Figure 1A of our previously published 2015 eLIFE paper.Mammalian NHE1 has conserved domains that are only present in Drosophila Nhe2, including conserved sequences in the transmembrane domains, a lysine/arginine-rich region (KR motif) that facilitates membrane localization through phosphatidylinositol 4,5-bisphosphate (Putney and Barber, 2003), a conserved binding motif for the calcineurin homologous protein CHP (Lin and Barber, 1996;Pang et al., 2001), a functionally conserved glutamic acid residue in the transmembrane domain that is essential for H+ efflux (E358 in DNhe2 and E266 in human NHE1) and consensus sites for phosphorylation by Akt and ATM/ATR kinases in the C-terminus of both proteins.
3. In Fig. 1A, the genotype of each panel should be labeled.The control fly contains the white gene (W) mutation W1118 (left panel) that generates white-eyes, whereas the eyes in the middle and right panels are shades of red.Are the genotypes of the GMRGAL4+ and GMR>DNhe2 flies derived from W1118 in order to make a valid comparison of eye size?More description of the different genotypes is warranted for those not familiar with fly genetics.Fig.
5) Figure Notes a. Fig 3 C should include all the groups in Fig 3A Minor.